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Background The conditioned medium from human dermal fibroblasts (dermal fibroblast‐conditioned medium; DFCM) contains a diverse array of secretory proteins, including growth factors and wound repair‐promoting proteins. Angiogenesis, a crucial process that facilitates the infiltration of inflammatory cells during wound repair, is induced by a hypoxic environment and inflammatory cytokines. Methods In this study, we conducted a comprehensive bioinformatic analysis of 337 proteins identified through proteomics analysis of DFCM. We specifically focused on 64 DFCM proteins with potential involvement in angiogenesis. These proteins were further classified based on their characteristics, and we conducted a detailed analysis of their protein–protein interactions. Results Gene Ontology protein classification categorized these 64 DFCM proteins into various classes, including metabolite interconversion enzymes (N = 11), protein modifying enzymes (N = 10), protein‐binding activity modulators (N = 9), cell adhesion molecules (N = 6), extracellular matrix proteins (N = 6), transfer/carrier proteins (N = 3), calcium‐binding proteins (N = 2), chaperones (N = 2), cytoskeletal proteins (N = 2), RNA metabolism proteins (N = 1), intercellular signal molecules (N = 1), transporters (N = 1), scaffold/adaptor proteins (N = 1), and unclassified proteins (N = 9). Furthermore, our protein–protein interaction network analysis of DFCM proteins revealed two distinct networks: one with medium confidence level interaction scores, consisting of 60 proteins with significant connections, and another at a high confidence level, comprising 52 proteins with significant interactions. Conclusions Our bioinformatic analysis highlights the presence of a multitude of secretory proteins in DFCM that form significant protein–protein interaction networks crucial for regulating angiogenesis. These findings underscore the critical roles played by DFCM proteins in various stages of angiogenesis during the wound repair process.

Background Human dermal fibroblasts secrete diverse proteins that regulate wound repair and tissue regeneration. Methods In this study, dermal fibroblast‐conditioned medium (DFCM) proteins potentially regulating nerve restoration were bioinformatically selected among the 337 protein lists identified by quantitative liquid chromatography‐tandem mass spectrometry. Using these proteins, protein–protein interaction network analysis was conducted. In addition, the roles of DFCM proteins were reviewed according to their protein classifications. Results Gene Ontology protein classification categorized these 57 DFCM proteins into various classes, including protein‐binding activity modulator (N = 11), cytoskeletal protein (N = 8), extracellular matrix protein (N = 6), metabolite interconversion enzyme (N = 5), chaperone (N = 4), scaffold/adapter protein (N = 4), calcium‐binding protein (N = 3), cell adhesion molecule (N = 2), intercellular signal molecule (N = 2), protein modifying enzyme (N = 2), transfer/carrier protein (N = 2), membrane traffic protein (N = 1), translational protein (N = 1), and unclassified proteins (N = 6). Further protein–protein interaction network analysis of 57 proteins revealed significant interactions among the proteins that varied according to the settings of confidence score. Conclusions Our bioinformatic analysis demonstrated that DFCM contains many secretory proteins that form significant protein–protein interaction networks crucial for regulating nerve restoration. These findings underscore DFCM proteins’ critical roles in various nerve restoration stages during the wound repair process.

In our study, we investigated the chemical composition and cytotoxic activity of essential oils isolated from Dalmatian sage (Salvia officinalis L.) collected along the Adriatic coast of Croatia. Scanning electron microscopy (SEM) was used to examine the morphology of the stem and leaf surfaces. Essential oil excretory glands were detected on both the leaves and stem surfaces. The essential oils were isolated by hydrodistillation, and their chemical composition was determined by gas chromatography and mass spectrometry (GC-MS). Sage essential oils were mixtures of terpene compounds, among which the most common were: α- and β-thujone, camphor, and 1,8-cineol. Cytotoxic activity was tested using MTS assay on multiple cell lines: normal and immortalized fibroblasts (HF77FA and HDF-Tert), immortalized lung line (BEAS-2B), and breast adenocarcinoma (MDA-MB-231). The growth of treated cells was determined relative to control conditions without treatment. The immortalized lung line was the least resistant to the activity of the essential oils, whereas immortalized fibroblasts were the most resistant. Statistical analysis has connected the cytotoxic effect and chemical composition of the studied essential oils. To the best of our knowledge, this work is the first testing of the cytotoxic activity of S. officinalis EO’s on the BEAS-2B, HF77FA, and HDF-Tert cell lines. The presented data on essential oil chemical composition and cytotoxic effect on 4 types of human cells supports pharmacotherapeutic potential this plant is known to have.

Helichrysum italicum (Roth) G. Don., immortelle, is a plant species used in ethnomedicine and the food industry as a spice added to food, beverages, and bakery products. It has been shown to possess various biological activities, such as antioxidant and antibacterial activity, making it useful as a natural preservative. We investigated the phytochemical profile and biological activity of H. italicum essential oils from wild-grown plant material collected from natural habitats in the Republic of Croatia and Bosnia and Herzegovina. Using high-resolution scanning electron microscopy (SEM), a visual investigation of plant organs (stem, leaf, and flower) was performed, confirming the presence of essential oil reservoirs on the surface of all examined plant organs. Essential oils were isolated by hydrodistillation in the Clevenger apparatus. The chemical composition of the essential oils was determined using the GC-MS analytical technique. Cytotoxic activity tests were performed in vitro on three cell lines: skin (fibroblast), lung, and breast cancer. Using statistical tools, the synergistic and selective effects of H. italicum essential oil on healthy and tumor cells were correlated to chemical composition and cytotoxic activity. The synergistic and antagonistic effects of H. italicum essential oil’s individual components were simulated by testing pure compounds and their mixture of cytotoxic activity on fibroblasts and breast cancer cells. The results confirm that essential oil’s biological activity is much greater than the sum of the effects of its components. The present data are novel contributions to the body of knowledge on the biological activity of this species used in the food industry.

Human dermal fibroblasts secrete numerous growth factors and proteins that have been suggested to promote wound repair and hair regeneration. Human dermal fibroblast-conditioned medium (DFCM) was prepared, and proteomic analysis was performed. Secretory proteins in DFCM were identified using 1-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis, in-gel trypsin protein digestion, and quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS). Identified proteins were analyzed using bioinformatic methods for the classification and evaluation of protein-protein interactions. Using LC-MS/MS, 337 proteins were identified in DFCM. Among them, 160 proteins were associated with wound repair, and 57 proteins were associated with hair regeneration. Protein-protein interaction network analysis of 160 DFCM proteins for wound repair at the highest confidence score (0.9) revealed that 110 proteins were grouped into seven distinctive interaction networks. Additionally, protein-protein interaction network analysis of 57 proteins for hair regeneration at the highest confidence score revealed that 29 proteins were grouped into five distinctive interaction networks. The identified DFCM proteins were associated with several pathways for wound repair and hair regeneration, including epidermal growth factor receptor, fibroblast growth factor, integrin, Wnt, cadherin, and transforming growth factor-β signaling pathways. DFCM contains numerous secretory proteins that comprise groups of protein-protein interaction networks that regulate wound repair and hair regeneration.

Background: Human fibroblast-derived multi-peptide factors (MPFs) promote wound repair by playing crucial roles in cell recruitment, adhesion, attachment, migration, and proliferation. Methods: Cultured human dermal fibroblasts (HDFs) were directly treated with non-contact low- and high-energy nitrogen plasma and further cultured in various conditioned media. Cell proliferation and wound-healing properties were evaluated. Results: In Opti-modified Eagle's medium + GlutaMAX culture, reduced HDF viability was observed 24 h after 2- J/pulse plasma treatment and 12 and 24 h after 3-J/pulse treatment. Meanwhile, in dermal fibroblast-conditioned medium (DFCM) containing MPF culture, reduced HDF viability was observed only 24 h after 3-J/pulse treatment. Under DFCM-MPF culture, the wound area percentage was significantly decreased after 12 and 24 h in untreated HDFs; at 9, 12, and 24 h after 1-J/pulse plasma treatment; at 3, 6, 9, 12, and 24 h after 2-J/pulse plasma treatment; and at 9, 12, and 24 h after 3-J/pulse plasma treatment. Greater migration of HDFs with or without plasma treatment was found in DFCM-MPFs than in other conditioned media. Conclusion: Low-energy nitrogen plasma treatment promotes HDF proliferation and wound repair. DFCM-MPFs enhanced cell proliferation and improved the wound healing properties of HDFs treated with low- and high-energy plasma. Keywords: human dermal fibroblasts, multi-peptide factors, dermal fibroblast-conditioned media, plasma, nitrogen, cell proliferation, wound healing, cell migration

Aging of skin is associated with environmental factors such as ultraviolet rays, air pollution, gravity, and genetic factors, all of which can lead to wrinkling of skin. Previous reports suggest that the wound repair is impaired by the aging process and strategies to manipulate the age-related wound healing are necessary in order to stimulate repair. Several traditional plant extracts are well-known for their properties of skin protection and care. Piper cambodianum P. Fourn. (PPF), a member of Piperacecae, is a plant found in Vietnam that might have therapeutic properties. Therefore, the effects of PPF stem and leaf extract on aging process were investigated in vitro and in vivo. PPF extract dissolved in methanol was investigated using Western blotting, real-time quantitative reverse transcription-polymerase chain reaction, flow cytometry, and cell wound-healing assays. We assessed the anti-aging effect of PPF in mouse using the wound-healing assay. The results were analyzed by Student's unpaired t-test; *P<0.05 and **P<0.01 were considered to indicate significant and highly significant values, respectively, compared with corresponding controls. PPF treatment demonstrated in vitro and in vivo anti-aging activity. Western blot analysis of PPF-treated normal human dermal fibroblast cells showed a dose-dependent increase in the expression of extracellular matrix genes such as collagen and elastin, but decreased expression of the aging gene matrix metalloproteinase-3. Quantitative polymerase chain reaction showed that PPF-treated cells displayed dose-dependent increase in messenger RNA expression levels of collagen, elastin, and hyaluronan synthase-2 and decreased expression levels of matrix metalloproteinase-1 aging gene. PPF treatment led to decreased production of reactive oxygen species in cells subjected to ultraviolet irradiation. Furthermore, PPF extract showed positive wound-healing effects in mice. This study demonstrated the anti-aging and wound-healing effects of PPF extract. Therefore, PPF extract represents a promising new therapeutic agent for anti-aging and wound-healing treatments.

Background：Shikonin is a major active chemical component extracted from Lithospermi Radix,an effective traditional herb in various types of wound healing.Shikonin can accelerate granulomatous tissue formation by the rat cotton pellet method and induce neovascularization in granulomatous tissue.The purpose of the study was to investigate its mechanism of action in human skin cells.Methods：MTS assay was used to measure cell growth.The collagen type Ⅰ （COL1） mRNA expression and procollagen type Ⅰ C-peptide （PIP） production were detected by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay,respectively.Immunofluorescence and western blot analyses were carried out to investigate nuclear factor-κB （NF-κB） signaling pathway.Cell-based proteasome activity assay was used to determine proteasome activity.Results：In this study,we found that 10 μmol/L shikonin stimulated the growth of normal human keratinocytes and 1 μmol/L shikonin promoted growth of human dermal fibroblasts.However,shikonin did not directly induce COLI mRNA expression and PIP production in dermal fibroblasts in vitro.In addition,1 μmol/L shikonin inhibited translocation of NF-κB p65 from cytoplasm to nucleus induced by tumor necrosis factor-α stimulation in dermal fibroblasts.Furthermore,shikonin inhibited chymotrypsin-like activity of proteasome and was associated with accumulation ofphosphorylated inhibitor κB-α in dermal fibroblasts.Conclusions：These results suggested that shikonin may promote wound healing via its cell growth promoting activity and suppress skin inflammation via inhibitory activity on proteasome.Thus,shikonin may be a potential therapeutic reagent both in wound healing and inflammatory skin diseases.

UV radiation and H2O2 are the primary factors that cause skin aging. Both trigger oxidative stress and cellular aging. It has been reported that deacetylase silent information regulator 1 (SIRT1), a longevity gene, enhances activation of NF-E2-related factor-2 (Nrf2), as well as its downstream key antioxidant gene hemeoxygenase-1 (HO-1), to protect cells against oxidative damage by deacetylating the transcription coactivator PPARγ coactivator-1α (PGC-1α). Galangin, a flavonoid, possesses anti-oxidative and anti-inflammatory potential. In the present study, we applied Ultraviolet B/H2O2-induced human dermal fibroblast damage as an in vitro model and UVB-induced photoaging of C57BL/6J nude mice as an in vivo model to investigate the underlying dermo-protective mechanisms of galangin. Our results indicated that galangin treatment attenuates H2O2/UVB-induced cell viability reduction, dermal aging, and SIRT1/PGC-1α/Nrf2 signaling activation. Furthermore, galangin treatment enhanced Nrf2 activation and nuclear accumulation, in addition to inhibiting Nrf2 degradation. Interestingly, upregulation of antioxidant response element luciferase activity following galangin treatment indicated the transcriptional activation of Nrf2. However, knockdown of SIRT1, PGC-1α, or Nrf2 by siRNA reversed the antioxidant and anti-aging effects of galangin. In vivo evidence further showed that galangin treatment, at doses of 12 and 24 mg/kg on the dorsal skin cells of nude mice resulted in considerably reduced UVB-induced epidermal hyperplasia and skin senescence, and promoted SIRT1/PGC-1α/Nrf2 signaling. Furthermore, enhanced nuclear localization of Nrf2 was observed in galangin-treated mice following UVB irradiation. In conclusion, our data indicated that galangin exerts anti-photoaging and antioxidant effects by promoting SIRT1/PGC-1α/Nrf2 signaling. Therefore, galangin is a potentially promising agent for cosmetic skin care products against UV-induced skin aging.

Evidence suggests that the increased production of free radicals and reactive oxygen species lead to cellular aging. One of the consequences is lipid peroxidation generating reactive aldehydic products, such as 4-hydroxynonenal (HNE) that modify proteins and form adducts with DNA bases. To prevent damage by HNE, it is metabolized. The primary metabolic products are the glutathione conjugate (GSH-HNE), the corresponding 4-hydroxynonenoic acid (HNA), and the alcohol 1,4-dihydroxynonene (DHN). Since HNE metabolism can potentially change during in vitro aging, cell cultures of primary human dermal fibroblasts from several donors were cultured until senescence. After different time points up to 30 min of incubation with 5 µM HNE, the extracellular medium was analyzed for metabolites via liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI-MS). The metabolites appeared in the extracellular medium 5 min after incubation followed by a time-dependent increase. But, the formation of GSH-HNL and GSH-DHN decreased with increasing in vitro age. As a consequence, the HNE levels in the cells increase and there is more protein modification observed. Furthermore, after 3 h of incubation with 5 µM HNE, younger cells showed less proliferative capacity, while in older cells slight increase in the mitotic index was noticed.